Pulmonary arterial hypertension (PAH; WHO Group I) is a disease of the small pulmonary arteries, characterized by vasoconstriction, vascular proliferation, and remodeling. Over the first 4 years of our translational Program Project Grant (tPPG), we have focused on the less studied sub-phenotypes of pulmonary hypertension, namely pulmonary venous hypertension (PVH or Group II), which typically arises in the setting of the metabolic syndrome. Group II PVH is also referred to as pulmonary hypertension in the setting of heart failure with preserved ejection fraction (PH-HFpEF) and is extremely common, with no currently approved effective therapies. It has become increasingly apparent that major risk factors for the development of both Group I disease and Group II (PH-HFpEF) include all features of the metabolic syndrome (obesity, insulin resistance, and systemic hypertension). Importantly, both the relaxation and abnormal proliferation of pulmonary vascular smooth muscle cells and the central mechanisms underlying the metabolic syndrome are strongly modulated by nitric oxide (NO)-dependent reactions, inducing both cGMP-dependent vasodilation and cGMP-independent reactions that inhibit smooth muscle cell proliferation, inflammation, and oxidative stress, as well as improve insulin sensitivity. In Project 1 it is hypothesized that new vascular-targeted, NO-based therapeutic strategies will enhance the treatment of PAH and metabolic syndrome, providing a new therapy for the currently untreatable and extremely common PH-HFpEF. To this end, we have identified two reactive nitrogen species that potently modulate both PH and the metabolic syndrome, nitrite (NO2-) and nitro-fatty acids (NO2-FA). In our preliminary data we find that nitrite (NO2-) can be bioactivated in skeletal muscle, via the myoglobin oxidoreductase reaction, to activate mitochondrial SIRT3- AMPK-Glut4 signaling pathways to improve glucose homeostasis and reverse established PH-HFpEF. Interestingly, nitrite also activates AMPK in the pulmonary vascular smooth muscle, independent of SIRT3 signaling, suggesting signaling convergence around AMPK. In addition to direct bioactivation of nitrite in smooth and skeletal muscle, nitrite reacts with dietary linoleic acid in the acidified stomach to generate nitrated fatty acids, which are potent electrophiles that post-translationally regulate Keap1-Nrf2 mediated antioxidant and anti-inflammatory signaling. We have now developed novel mouse and rat models of PH-HFpEF and have completed critical human phase 2 safety and proof of concept clinical trials of oral and inhaled nitrite therapy in patients with PAH. For the next phase of support we plan to study the mechanisms underlying the development of PH-HFpEF in the setting of metabolic syndrome. Fulfilling the translational directive of the tPPG, our project extends our completed phase 1 PK and safety studies to a randomized placebo-controlled trial of oral nitrite versus placebo for patients with PH-HFpEF, for which there is no currently approved therapy.